Background:  Efavirenz (EFV) has recently been associated with changes in lipid and body fat composition characteristic of lipodystrophy. In this study we have analyzed the molecular mechanisms that could be implicated in these alterations of lipid metabolism, and the role of the master switch of the regulation of cellular bioenergetics, AMP-activated protein kinase (AMPk).

Methods:  Non-infected Hep3B cells were stained with the fluorescent probe Nile Red after 24-h incubation with EFV (10, 25, or 50 mM) and measured by static cytometry. In order to define the nature of these accumulated lipids, cells were treated for 4 h with EFV (10 and 25 mM) and intracellular lipid content was determined by nuclear magnetic resonance (NMR). The expression of the fatty acid transporters CD36 and FATP was analyzed by semi-quantitative polymerase chain reaction (PCR). Selected experiments were performed in cells pretreated (30 minutes) with the AMPK inhibitor compound C (20 mM). Data (n ≥3) were analyzed by ANOVA (1-way or repeated measures).

Results:  EFV induced neutral lipid accumulation in hepatic cells after 24-h incubation (10 mM = 124.2±3.2%, 25 mM = 125.6±7.6%, 50 mM = 147.1±9.8% vs control 106.1±1.3%). In NMR experiments, the effects of EFV were also significantly evident following 4-h incubation (values of relative intensity signal in arbitrary units; 10 mM = 0.1154±0.007 arbitrary units, 25 mM = 0.1126±0.0082 arbitrary units vs control = 0.097±0.0112 arbitrary units), and the profile of the lipids suggested that they did not originate in the membranes. Alteration of lipids by EFV was not observed when fatty acids were removed from the culture media, thus demonstrating that they were the result of fatty acid uptake from extracellular sources. Likewise, these changes did not occur in the presence of compound C, pointing to a key role of AMPK in this lipid uptake. This enzyme was also implicated by the fact that EFV increased CD36 and FATP mRNA expression, 2 of its downstream targets.

Conclusions:  Our results demonstrate that EFV increases fatty acid uptake in hepatic cells, leading to the accumulation of lipids. These lipids have the same chemical composition as those that form droplets. Given the long-term treatment of patients with EFV, such effects on lipid content suggest that this drug exerts a pro-steatotic role in the liver and could alter lipid metabolism in this organ. Due to the importance of the liver in the general regulation of lipids, EFV may also be implicated in some of the alterations that are characteristic of lipodystrophy.